1 rudolf virchow, die cellularpathologie (1858) all diseases involve changes of cells all cells come...
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Rudolf Virchow, Die Cellularpathologie (1858)
All diseases involve changes of cells
All cells come from cells
Lecture 6 & 7:Cell biology 2014 (revised 4/2 -14)
Cells of all life forms have a common ancestor
1013
Controlled and co-ordinated divisions
somatic mutations &chromosomal instability
Cell cycle control in multi-cellular eukaryotes
Uncontrolled divisions Tumor (clonal origin!)
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Human diploid genome: ~6 x109 bp (cost of MD education: ~6 x109 Skr/year)(length of 1 bp) x (number of bp per cell)
(0.34 nm) x (6 × 109) = 2 m, 1013 cells 2 x 1013 m
0.5 x1013 m
Earth Uranus Pluto
SunMars
Minimal length of DNA replicated by (cells of) a human
“Simple” model systems for studies of eukaryotesBudding yeast, S. cerevisiae Polarized (bud & shmoo)
Fission yeast, S pombeSymmetric cell division
Yeast model systems:
- Unicellular eukaryotic organism (autonomous cells)- Sexually active (mating & sporulation)- Short generation time - Haploid: phenotype of recessive (loss-of-function) mutations
vs.
3
Primordial eukaryote(polarity & sex)
The mutant gene product is temperature sensitive (Ts)OK at 26oC, but unfolds at 36oC
(i.e. the function of gene product can be switched off)
Wild type protein
Functional(i.e. no phenotype)
26oC
36oC Non-functional(i.e. phenotype)
Principles of conditional mutants
4
Mutant protein
26o C
36o C
Missing!
Cell cycle Ts mutant or not?
26o C
House keepinggene mutated
Cell cycle controlgene mutated
36o C
Temperature sensitive yeast mutants 5
or(if lucky!)
= Ts clone
36o C+
Gene library(i.e., wild type genes cloned
into bacterial plasmids)
Yeast cellcycle Ts mutantMutated gene:(OK at 26oC but not 36oC)
Identification of cell division control (cdc) genes
ZZZ
ZZZZZZ
ZZZ
6
OK
OK
Complementation by : OK
Cell cycle regulators are evolutionary conserved Budding yeast with a temperaturesensitive mutation in an essential cell division control (cdc) gene
cDNA library (copies of all human mRNA’s) identification of a human “ortholog”
The cell cycle control machinery is highly conserved in eukaryotsNobel prize in medicine 2001!
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36o C
36o C
Three distinct cell cycle regulated events
Increased size
Nuclear division followed by cytoplasmic division
DNA replication
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G1 (Gap 1)
G2 (Gap 2)
S (DNA-Synthesis)
Cell cycle exit “G0”
Interphase (“between-phase(s)”)(90100% of a cell population)
Mitosis (Cell division phase)(010% of a cell population)
Chromosome segregation
Cell division
9
Quiescent orpost-mitotic
Condensed chromatin”thread-like”
Interphase and mitosis
Chromatin“attract dyes”
G2
S
G1
GO
(Greek: mitos= thread, khrōma = color, soma = body)
Key events and checkpoints of the cell cycle
G2/M checkpoint
Spindle assembly checkpoint
G1/S checkpoint (“Start”)
G1
SG2
M1.
2.
3.
1.
2.
3.
Sense: Surroundings Cell size
Block: DNA replication
Sense: DNA replication status DNA damage Cell sizeBlock: Mitotic entry
Sense: Chromosome attachment to the mitotic spindleBlock: Chromosome separation and cytoplasmic division 10
Cell cycle regulation of proteinsAmount of protein
- Control of protein expression
Protein
DNA
mRNATranscription
Translation
- Control of protein turnover
Ub
Ub
Ub
26S Proteosome
Activity of protein
- Binding partners
+ =
+ =
- Phosphorylation
PP
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P
Cyclin dependent kinases (Cdk) – the controllers
O
Serine or threonine
Serine or threonine
O
O
O -P
Kinase
O
-
H
P
Cdk Cyclin
Kinase motifInhibitory domain (T-loop)
P
Cyclin substrate specificity
Both the cyclin and are requiredto activate the Cdk
P
Phosphatase
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G1/S(E)
S(A)
G1(D)
M(B)
G1Diffuse border
G2 M
Cyclin:(expression)
Cdk 2
Cdk 4/6
Cdk 1
Cdk’s are stable while cyclin levels are “cyclic”
13
Ras v-SNARE
NFAT
Hsp
70
Clathrin
COPII MHCACTH
cGM
P
Retinoic acid
Src R7
Notch
Wnt
Ci
RelA
cGMP
cGM
P
v-SNARE
v-SNARE
v-SNAREv-SNARE
The cell can not reside in two cell cycle phases simultaneously
The cell can not reside in two cell cycle phases simultaneously
G1M S
Interphase cyclins Mitotic cyclin
Cdk S
Mechanisms for interphase cyclin degradation
Cdk G1/S
Cdk S
Cdk G1
Cdk M
1.
2.
3.
High intrinsicturnover of G1 cyclin
Cdk +
-dependent ubiquitination of G1/S cyclin
P
Cdk G1/S
P
Ub
UbUb
Cdk +
PUb
UbUb
P P
Ub
UbUb
+
PUb
UbUb
Cdk
Cdk/M-cyclinmediated ubiquitinationof S cyclin
Kinase X
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P
P
G1 S G2 M
Cdk G1/S
Cdk G1
Cdk M
G0
S Cdk
15
Transitions:
Cdk/cyclin control of progression and transitions
G1M
Cdk S Active: Cdk X
External signals (mitogens)G1 cyclin expression
Wnt
Receptor Tyrosine Kinase
Hedgehog
G1
G1 cyclin gene
MycMyc
myc gene
XGF
GTPRas
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= DNA
E2F
Rb
E2F
repressor activator
Cdk G1/S
DNAreplisome
Cdk G1 G1/S
S
1. 2.
2.
Phosphorylation by Cdk-G1 cyclin dissociates Rb from E2F 1.
Positive feedback loop
RbP P
Rb phosphorylation by Cdk/G1 cyclin E2F activation
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3.
Enhancement by newly formed Cdk-G1/S cyclin3.
Transcription of E2F regulated genes
= DNA binding protein
DNAreplisome
E2F
Proliferating cell:E2F mediated transcription of “S phase genes”
E2F
Rb
Non-dividing cell:Dominant repression by the E2F/Rb complex
Transcriptional control of S phase components
Rb is mutated in ~40% of human tumors constitutive production of S-phase components
S-phase componentsS
G1/S Rb
P P
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E2F
S-phase components
E2FE2F
The stability of the genome: two levels of threat
G2
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S-phase: 46 x ~4 cm DNA has to be replicated once (but only once!)
S-phase(6 h) ~4 cm DNA= 130 x106 bp
~ 250 nt/s 144 h/ 4 cm
G1
M-phase: sorting of 2 x 46 sister chromatids (no errors!)
G1
Licensing of DNA for a single round of replication
Early G1 ORC
ORCCdc6
Formation of a pre-RC (i.e., licensing of DNA): Cdc6 dependent loading of Mcm proteins onto DNA
Cdc6 is recruited to ORCs
Note - pre-replicative complexes are formed after mitosis independently of progression into a new S-phase
ORC ORC
ORCCdc6
ORCCdc6
ORCMcm
Mcm
Cdc6ORCM
cm
Mcm
Cdc6
ORCMcm
Mcm
Cdc6
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DNAreplisome
Cdk S
Firing of pre-RC during S-phase
DNAreplisome
DNAreplisome
Firing of the first ORC point of no return!
Late G1 ORCMcm
Mcm
Cdc6
S Mcm
Mcm
ORC
Cdc6
ORC
P
DNA strand separation by helicase activity of Mcm proteins
Phosphorylates Cdc6 and Mcm
P P
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A fool-proof system for prevention of re-replication
2.
3.
Cdc6P
Cdc6P
S CFUb
UbUb 4.
2.
3.
1.
4.
Phosphorylated Cdc6dissociates from ORC
Phosphorylated Cdc6 isrecognized by SCF
Ubiquitylation of Cdc6by SCF (an E3-ligase)
ProteosomeProteosomal degradationof Cdc6
DNAreplisome
DNAreplisomeM
cm
Mcm
ORC
ORC1.
P P
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Cdc6
Prevention of DNA licensing until the next G1-phase
G1S G2 M
Cdc
6 le
vels
PCdc6
Ub
UbUb
Cdk S
PCdc6
Ub
UbUb
PCdc6
Ub
UbUb
Cdk MS Cdk
Cdc6
ORCMcm
Cdc6
Degradation of “free” Cdc6G1: “free” Cdc6 is
available
Cdc6Cdc6
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Cdk G1
Cdk S
Rb
E2F
Cell cycle entry and DNA replication
Cdk G1/S
MycGrowth factor G1
External signal Activation of the Rb pathway!
DNAreplisome
Mcm
ORCCdc6
P
P
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Positive feedback loop
Rb/E2F = dominant repressor of E2F
p53: The guard against tumors
p53
Mdm2
Ub
UbUb
p53
Transcriptional activation of p53 responsive genes
2.3.
4.
1.
2.
3.
4.
DNA damage ( ) or unbalanced/excessive proliferation signaling ( ) inhibits Mdm2 stabilization of p53
degraded through ubiquitination by Mdm2 (E3-ligase)
p53 is constitutively expressed but is normally…
p53p53ATM
ARF
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p531.
p53 gene
3.
ATM
ARF
(ATM: ataxia telangiectasia-mutated, ARF: Alternative Reading Frame)
Target genes of p53 dependent transcription
p21DNA repairproteins
Cell cycle block DNA repair Apoptosis
Loss of p53 function
I´m gonna live foreverGenetic instability
p53
PUMABax
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Two distinct CdK Inhibitor (CKI) families
p
16
Cyclin
p21
CdkG1
CyclinCdk 4/6
Inhibits: Catalytic activity Cyclin association (Cip/Kip family) (Ink4 family)
Specificity: G1, G1/S and S G1 only
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In both cases, the Cdk activity is abolished
Molecular_models: 17.1-Cdk2
Cdk G1
Cdk S
Rb
DNAreplisome ORC
Cdc6P
E2F
p16
Rb & p53 pathways cell cycle and apoptosis
p53
Cdk G1/S
Myc
p53 pathway detects:1. Unbalanced/ excessive proliferation signals (+++)2. DNA damage
Cell cycle block Apoptosis
ARF
ATM
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PI3K
Apoptosisp21
G1/S block
PK
B/A
kt
Survivalsignals
++
+
+++
Growth factor
Ras
1.
2.
1.
Three distinct cell cycle regulated events
Size growth
Nuclear division followed by cytoplasmic division
DNA replication
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Cell division and cell growth
Oocytes grow without dividing
Fertilized eggs replicate and divide without growing30
PI3-kinase signaling regulates protein synthesis
P.M.
P
PP
P
PP
PDK1 PKB/Akt
3 3
PKB/AktP
P
PKB/ Akt
Ribosomes
Translation initiation factor
= ProteinsmRNA+ Cellular size growth
+31
RTK
P
PI-3 KP
Divergent (cooperating) RTK receptor signals
P3 3 P
PP
P
PKB/AktP
RTK
P
PI-3 K
Ras PTEN
Survival
Bad
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”Mitogen” signaling(Rb-pathway)
”Growth factor” signaling
PPP
Increasedcell size
Cell cycleentry
G1 G1
G1
DNA replication
Summary: G1-, S- and G2-phase (interphase)
G1 G1 G1 G1
G1 G1 G1
G1 G1
G1
DNA replisome
”Mitogen” signaling:
Size growth
+ =Translation
Initiation factor”Growth factor” signaling:
Bad
Ras
PI-3 K33
The end of the cell cycle
Cell division = nuclear division + cytoplasmic division
M-phase Mitosis Cytokinesis
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Sorting of 2 x 46 sister chromatids
Defines the division plane/symmetryProtein sorting (if asymmetric division)
Topics of case 13: The cytoskeleton
Progression of cell division: two points of “No return”
Prometaphase
MetaphaseAnaphase
Telophase/cytokinesis
ProphaseInterphase (G2)
Transition points Checkpoint control
Video: 17.4Animal_cell_division
35
G2 M transition
MAPsP
Mitotic spindle formation
Event Cause
Change in MT dynamics
CondensinP
Chromosome condensation DNA packaging
Nuclear laminsP Breakdown of
nuclear envelopeDisassembly of nuclear lamina
Consequence
36
Checkpoint control of Cdk/M-cyclin activity
+ Cdk MCdk M Cdk M Cdk MP P
P Cdk MP
wee1 CAK Cdc25
Inactive Inactive Inactive Active
G2 G2M
PP
P
Inhibitory sites:Activating site:
P P
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Dual feedback loops activity burst!
Cdc25DNA damageDNA replication stall
G2/M checkpoint control and regulation of Cdc25 activity:
Insufficient cell size
Chromosome segregation
Ub
UbUb
+
Ub
UbUb
Cohesin Separase Securin
AttachedduringS-phase
+
Sister chromatid separation (anaphase)
Anaphase initiation
38
Checkpoint control of metaphase-anaphase transition
APC/C
Cdc20 Active
APC/C
Inactive
1 unattached kinetochore(spindle assembly checkpoint)
Cdc20
+
Ub
UbUb
Metaphase Anaphase39
Sorting of 2 x 46 chromosomes
Mitotic exit and cytokinesis
Mitotic exit and initiation of cytokinesis
40
Cdk MActive P
Cdk MInactivation of
Ub
UbUb
Checkpoint
Cdk/M activity block initiation of cytokinesis!
Molecular events during mitotic exit
APC/C
Cdc20
+
Ub
UbUb
Cdk M Cdk M
Ub
UbUb
Cdk
Active APC/C cyclin degradation inactive Cdk mitotic exit
CondensinP
MAPs
Nuclear laminsP
P
Mitotic exit
Condensin
Nuclear lamins
MAPs
Constitutively active phosphatases drives the cell out of mitosis
Phosphatase
41
Mitosis versus meiosis:Animation: 21.2-meiosis
Ub
UbUb
+
P P
Cdc25
G1
SG2
MG2/M checkpointDNA status and cell size
Spindle assembly checkpointSpindle attachment to chromosomes
G1/S checkpointThe surroundings
APC/C
Cdc20
Checkpoints function: block of premature transitions
DNAreplisome
Mcm
Mitogen
PCdk M Cdk MP PPP
E2F
42
Checkpoints verify that all processes at each cell cycle phase have been completed before transition into the next
Cell death in multi-cellular eukaryots
Cell death
Necrosis Apoptosis
- ”Murder”- Associated with inflammation (and vice versa)
- ”Suicide”- Death for the benefit of the organism- No inflammatory response
The word apoptosis is greek for fallen leaves
43
Development Homeostasis Immune survelliance
x x
Importance of apoptosis
Apoptosis and cell proliferation must balance each other
Killing of virus infected cells
Elimination ofsuperfluous cells
between the developing fingers
44
Cellular changes during apoptosis
Normal cell Different stages of apoptosis
Cell shrinkage
Membrane ruffling
DNA condensation
DNA fragmentation
Phagocytic cells recognizes phosphatidyl serine ( ) which becomes exposed on the surface during apoptosis
Video: 18.1 Apoptosis45
Burp!
Apoptosis – a protease cascade
DNA
Cytoskeleton
Other things
AD
N
Cy t
osk
Active effector caspase
Inactive effector caspase
Active initiator caspase
Initiatorcaspase
monomers
46
Apoptotic signal
multimers
=
Control of apoptosis
Anti-apoptoticPro-apoptotic Cell survival
Apoptosis
External and internal signals
• Two pathways for initiation of apoptosis: Extrinsic (receptor-mediated) and Intrinsic (mitochondrial)
• Apoptosis is controlled by the balance of Pro- and Anti-apoptotic regulatory proteins
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The intrinsic apoptotic pathway
Cyt. C
Cyt. C Cyt. C
Cyt. C
Bcl-2 BH123
Cyt. C
Pro-apoptotic
Pore formation in the outer mitochondrion membrane
release
Pore former Inhibitor of pore formation
48
Note - Bcl2 has all four BH domains(Albert et al: Fig. 18-9)
Anti-apoptotic
Cyt. C
Cytochrome C in the cytosol triggers apoptosis
Caspase 3
Caspase 9Apaf1
Cyt. C
Apaf1Cyt. C
Caspase 9
Apaf1Cyt. C
Caspase 9
Caspase 3
Apoptosome
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Target proteins
a g t r t i sT r e p o e n
: APoptosis Activating Factor
Regulation of the intrinsic apoptotic pathway
Bcl-2 BH123 (family)
Pro-apoptotic
BH3-only (family)
Bax
50
Note - Bcl2 has all four BH domains per definition! (Albert et al: Fig. 18-9)
PUMA p53
Anti-apoptotic
ARFATM
p53 pathway: Pro-apoptotic downstream mediators
BH3-only
Effector caspases
- DNA- Cytoskeleton- Other things
AD
N
Cy t
os
k
Survival signals Death
signalsCyt. C
Deathreceptor
Ligand
Extrinsic apoptosisPlasma membrane
Summary on apoptosis
Intrinsic apoptosis
BH123Bcl-2
Initiator caspaseInitiator caspase
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Determinants of survival and proliferation
Pro-apoptotic
G1 cyclin CKI
Regulation of cell survival Regulation of cell proliferation
+
x x
or
+
+
Apoptosis
Cell proliferation
Quiescent cell (G0)
or
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Anti-apoptotic
A single external signal may serve multiple functions
Growth factor
Survival factorMitogen
Anti-apoptotic
Pro-apoptotic
G1 cyclin +
+
G1
One specific ligand/receptor complex may transduce several distinct signals
Ribosome Translation initiation
factor
53
Translation Initiation
factor
Recommended reading
Alberts et al5th edition
Chapter 17
1053-10801092-10941101-1112
Chapter 18
1115-1128
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